Anti-tuberculosis activity of morusin: a promising flavonoid from white mulberry.

BACKGROUND: TB has remained a significant public health concern from historical times to the present day. Each year, growing drug resistance problems necessitate the discovery of new drugs and drug precursors for TB treatment. Morusin is an important flavone found in the bark of white mulberry (Morus alba L.) with anti-oxidant, antimicrobial, anti-tumour, anti-inflammatory and antiallergic activity.OBJECTIVE: To determine the anti-TB efficacy of morusin on Mycobacterium tuberculosis strains.DESIGN: Anti-TB efficacy of morusin was tested on H37Ra (American Type Culture Collection [ATCC] 25177), H37Rv (ATCC 27294), ATCC 35822 (isoniazid [INH] resistant), ATCC 35838 (rifampicin [RIF] resistant), and ATCC 35820 (streptomycin [SM] resistant) standard strains and its efficacy was determined using nitrate reductase assay (NRA).RESULTS: The minimum inhibitory concentration (MIC) of morusin was tested in the range of 53.83â-"0.21 λg/ml. The MIC for H37Ra (ATCC 25177), H37Rv (ATCC 27294) and ATCC 35838 (RIF-resistant) strains were found to be 6.72 λg/ml, and this was 13.45 λg/ml for the ATCC 35822 (INHresistant) and ATCC 35820 (SM-resistant) strains.CONCLUSION: To consider morusin as a viable alternative or precursor drug for TB treatment, it is imperative to conduct an exhaustive examination of its mechanism of action and conduct in vitro studies using clinical isolates.

Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates.

AIMS: To investigate plasmid-mediated quinolone resistance in clinical isolates of Pseudomonas aeruginosa with the polymerase chain reaction (PCR). The plasmid-mediated quinolone resistance genes have been identified in many bacteria within the Enterobactericeae family, they have not been detected in P. aeruginosa isolates. Subjects and Methods : Identification of the isolates and testing of antibiotic susceptibility was performed in Vitek2 Compact (Biomeriux, France) and Phoinex (BD, USA) automated systems. Screening for the qnrA, qnrB, qnrS, qnrC, aac (6')-Ib-cr and qepA genes was carried out by PCR amplification and aac (6')-Ib-cr DNA sequencing. RESULTS: The qnr and the qepA genes were not detected in any of P. aeruginosa isolates. The aac (6')-Ib gene was detected in six of the isolates and positive isolates for aac (6')-Ib were sequenced for detection of the aac (6')-Ib-cr variant but aac (6')-Ib-cr was not detected in any isolates. CONCLUSIONS: Plasmid-mediated quinolone resistance genes have so far not been identified in P. aeruginosa isolates. However, qnrB have detected in P. florescens and P. putida isolates. This is the first study conducted on the qnrA, qnrB, qnrS and qnrC genes as well as the qepA and aac (6')-Ib-cr genes in P. aeruginosa clinical isolates.

Human leucocyte antigens and cytokine gene polymorphisms and tuberculosis.

PURPOSE: Several genes encoding different cytokines and human leucocyte antigens (HLA) may play crucial roles in host susceptibility to tuberculosis (TB). Our objective was to investigate whether these genes might be associated with protection from or susceptibility to TB. MATERIALS AND METHODS: Genomic DNA from patients with TB (n = 30) and ethnically matched controls (n = 30) was genotyped by using sequence-specific primers-polymerase chain reaction and sequence-specific oligonucletid methods. RESULTS: Our results demonstrated that HLA-CwFNx0101 [P = 0.05, odds ration (OR) (95% confidence interval) = 2.269 (1.702-3.027)] allele frequency was significantly more common in TB patients than in healthy controls, and HLA-CwFNx0101 may be associated with susceptibility to TB. Analysis of cytokine allele frequencies showed that interleukin (IL)-10, -819 C and -592 C alleles was significantly more common in TB patients than in controls (pc: 0.038 and 0.017, respectively). From the IL-10 cluster, a positive significant difference was found at positions -1082 and -592 C/C (pc: 0.027 and 0.054, respectively) genotypes. Although these differences could be explained by the highest frequency of C/C and G/G homozygous patients with TB, in contrast to the control group, statistically significant differences for the C/C genotype however were lost after Bonferroni correction of the P-values. CONCLUSION: Altogether, our results suggest that the polymorphisms in HLA (class I) and cytokine (IL-10) genes may affect the susceptibility to TB and increase the risk of developing the disease.

Serum galectin-3 levels in patients with Behçet's disease: association with disease activity over a long-term follow-up.

BACKGROUND: There is a need for a laboratory marker that correlates with the clinical activity of Behçet's disease (BD). OBJECTIVE: We aimed to investigate whether serum galectin-3 (Gal-3) levels were affected during the course of the disease with regard to disease activity. METHODS: A total of 131 subjects were involved in the study as follows: Group 1: BD active (n = 39); Group 2: BD inactive (n = 31); Group 3: Disease controls with leucocytoclastic vasculitis confirmed with a skin biopsy (n = 22); and Group 4: Healthy control subjects (n = 39). The BD patients were followed regularly and samples were taken in their active and inactive periods of the disease over a 2-year period. RESULTS: Serum Gal-3 levels were significantly higher in active BD patients (mean 2.38) than inactive BD patients (mean 0.63; P < 0.0001) and the healthy control subjects (mean 0.75; P < 0.0001). There was no significant difference between the leucocytoclastic vasculitis and active BD patients (P = 0.093). Serum Gal-3 levels were positively correlated with clinical activity scores of active BD patients (r = 0.66, P < 0.0001). In addition, the Gal-3 levels were significantly higher in the active disease period when compared with the inactive period during the follow-up. There were no significant differences between the two inactive periods of the disease among the same patients. Further analyses revealed that patients with vascular involvement had significantly higher Gal-3 levels than the other active BD patients (mean 7.57; P = 0.007). LIMITATIONS: The limitation of the study is the small number of patients with vascular involvement in the active BD patient group. CONCLUSION: Gal-3 levels are correlated with the activity of Behçet's disease especially with the vascular involvement.

The effect of tigecycline and ertapenem against clinical isolates of Brucella melitensis detected by E-test on different media.

In this study, in vitro activity of tigecycline (TIG) and ertapenem (ERT) against clinical isolates of Brucella melitensis and the effect of different media on in vitro test results were investigated. The in vitro effects of TIG and ERT to 38 B. melitensis isolates were comparatively investigated in brucella agar and 5% sheep blood agar. MIC value of ERT was 0.032 µg/mL in 23 of 38 and 20 of 38 isolates on blood and brucella agar, respectively. Minimum inhibitory concentration values of TIG were substantially different ranging between 0.064-0.25 µg/mL on blood agar. However, MIC values of TIG were similar on brucella agar with 0.25 µg/mL in 15 of 38 isolates and 0.5 µg/mL in 10 of 38 isolates. In conclusion, although ERT and TIG were effective against B. melitensis isolates in vitro, further studies are needed in order to determine the use of these novel drugs in treatment of brucellosis.

Rapid susceptibility test for Mycobacterium tuberculosis to isoniazid and rifampin with resazurin method in screw-cap tubes.

Multi-drug resistant (MDR) Mycobacterium tuberculosis is still a serious public health problem all over the world. MDR tuberculosis (MDR-TB) caused by these strains has emerged within the last decade and rapid detection is critical for the effective treatment of patients. Recently, a resazurin microtiter assay plate for detecting MDR strains was developed. In this study, it was adapted to screw-cap tubes and the activity of isoniazid (INH) and rifampin (RIF) to 50 M. tuberculosis clinical isolates was tested by this method for the first time. Results were compared with the radiometric reference method for the susceptibility testing of M. tuberculosis complex. The results of both methods were in 100% and 96% agreement for RIF and INH, respectively. Specificity, sensitivity, positive predictive value and negative predictive value were 91.7%, 100%, 92.8% and 100% for INH, respectively. All of these values were 100% for RIF. Susceptibility testing results were obtained on the 8th day of incubation for 42 isolates and on the 9th day for the other eight strains. Our results indicate that this method is suitable for the early determination of INH and RIF resistance in developing countries because it is inexpensive, rapid and easy to perform.

Blood agar for susceptibility testing of Mycobacterium tuberculosis against first-line drugs.

OBJECTIVE: To evaluate the performance of blood agar for the susceptibility testing of 50 Mycobacterium tuberculosis clinical isolates against isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB). DESIGN: The activity of the drugs was determined by the proportion method on blood agar instead of Middlebrook 7H10 agar according to Clinical Laboratory Standard Institute recommendations. The final concentrations of INH, RMP, SM and EMB were 0.2 microg/ml, 1 microg/ ml, 2 microg/ml and 5 microg/ml, respectively. RESULTS: The results were compared with the radiometric proportion method as the reference, and the agreements were determined as 100% for INH and RMP, 92% for SM and 96% for EMB. The specificity, sensitivity, positive predictive value and negative predictive value were 90.4% and 97.5%, 100% and 90%, 66.6% and 90% and 100% and 97.5% for SM and EMB, respectively, while these values were 100% for INH and RMP. The results of susceptibility testing were obtained on the 14th day of incubation. CONCLUSION: According to this preliminary study, our results suggest that blood agar can be used as an alternative medium for the susceptibility testing of M. tuberculosis strains against INH, RMP, SM and EMB in resource-limited countries. However, further studies are needed before implementating the method in diagnostic laboratories.

The resazurin microplate method for rapid detection of vancomycin resistance in enterococci.

Vancomycin-resistant enterococci (VRE) are a serious challenge for physicians because of the limited treatment options for infections caused by this organism. Prevention of VRE transmission in hospitals requires early detection of infected or colonized patients. Therefore rapid and correct detection of vancomycin resistance is essential. In this study, we use the resazurin microplate method (RMM), which is a modification of the NCCLS and BSAC broth microdilution methods to rapidly determine the susceptibilities of clinical enterococci isolates to vancomycin. The alteration in the RMM was relevant to the final bacterial count. In this method, inoculum that was 10-fold higher than standard methods was used. A total of 80 enterococci, including 11 VRE isolates and 6 vancomycin intermediate isolates, were screened with this modified colorimetric broth microdilution method. After 4 h of incubation 30 microl of 0.01% resazurin solution were added to each well and the plates were reincubated for color change for 5-10 min. The MICs were obtained at the 4th h. The results were in exact agreement with the NCCLS and the BSAC microdilution methods. Absolute and essential agreements were 100% and there were no minor, major or very major errors. In conclusion, this modified colorimetric broth microdilution method can be used as a reliable, easy, cheap and rapid method for early detection of VRE. Moreover, this method has the potential of being used to test the susceptibilities of different bacteria to other antibiotics.

Effects of acetyl salicylate and ibuprofen on fluoroquinolone MICs on Salmonella enterica serovar typhimurium in vitro.

In this study, the effects of acetylsalicylate and ibuprofen at 2, 4 and 8 mM concentration were investigated on ofloxacin, ciprofloxacin, levofloxacin and pefloxacin minimum inhibitory concentrations (MICs) for 14 Salmonella enterica serovar typhimurium clinical isolates, one standard strain (SZH KUEN 557), SH7616 (acr mutant), SH5014 (parent strain of acr mutant) and PP120 (soxRS mutant) strains. All isolates were susceptible to the 4 fluoroquinolones. In the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen, 2- to 8-fold increases were observed in fluoroquinolone MICs. This rise was higher, especially in the presence of acetylsalicylate. In spite of this rise, none of the MICs were in the range of resistance limits in vitro. Except for a 2-fold increase in levofloxacin MICs, we did not observe any difference in MICs of ofloxacin, ciprofloxacin, and pefloxacin in the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen for SH7616 and PP120 strains. According to the in vitro results of this study, it can be suggested that use of acetylsalicylate or ibuprofen together with clinical treatment of bacteria, especially bacteria which show intermediate resistance, will cause resistance. However, since clinical data are insufficient, further studies are needed.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro.

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance

In vitro effects of cefotaxime and ceftriaxone on Salmonella typhi within human monocyte-derived macrophages.

The main objective of this in vitro study was to assess the effects of cefotaxime and ceftriaxone in killing Salmonella typhi in infected human macrophages. Human monocyte-derived macrophages isolated from peripheral blood of human volunteers were cultured in vitro for macrophage differentiation, and subsequently infected with S. typhi strains (a clinical isolate and a standard strain TA-42) at a cell ratio of 10 : 1. MICs of cefotaxime and ceftriaxone were determined by broth microdilution, and the antibiotics were included in the culture medium at one and five times their MIC values. Samples of cell culture medium taken at 0, 3, 6 and 24 h of incubation were cultured for growth of S. typhi on nutrient agar. Gentamicin (10 mg/L) was included in each well except for the control wells, in order to prevent growth of extracellular S. typhi. Both antibiotics showed good in vitro antibacterial effects against S. typhi strains. There were no statistically significant differences between the extracellular and intracellular effects of antibiotics with regard to elimination of the bacteria. Cefotaxime and ceftriaxone are highly effective against extracellular bacterial growth. The results of our in vitro experiments suggest that cefotaxime and ceftriaxone might also be used clinically against susceptible intracellular pathogens such as S. typhi.

A comparison of the E-test and proportion methods for susceptibility testing of Mycobacterium tuberculosis.

The aim of this study was to investigate the correlation between three antibiotic susceptibility methods, the proportion method on Löwenstein Jensen medium (LJ medium), the proportion method on Middlebrook 7H11 agar (7H11 agar), and the E-test for Mycobacterium tuberculosis. Fifty M. tuberculosis isolates were tested in vitro against isoniazid, rifampicin, streptomycin, and ethambutol according to the E-test, the proportion methods on 7H11 agar and LJ medium and then compared with a reference test which was the proportion method on 7H11 agar. The correlations between proportion method on 7H11 agar and proportion method on LJ medium for isoniazid, rifampicin, streptomycin, and ethambutol were 93.9%, 85.1%, 85.4% and 78.7% respectively. The correlations between the proportion method on 7H11 agar and the E-test were 83.1%, 78.8%, 84.7% and 80.5% respectively. There were no significant differences observed between the E-test and LJ medium compared to 7H11 agar. The average times to obtain susceptibility test results were 7 and 21 days for the E-test and agar proportion methods, respectively. The E-test may be suitable for replacing the proportion methods (7H11 agar and LJ medium) in routine practice due to its fast and easy application.